Technical Field
The present invention relates generally to methods and systems for analyzing specimens using energy. More specifically, the invention is related to methods and systems for analyzing tissue specimens using acoustic energy.
Description of the Related Art
Preservation of tissues from surgical procedures is currently a topic of great importance. Currently, there are no standard procedures for fixing tissues and this lack of organization leads to a variety of staining issues both with primary and advanced stains. The first step after removal of a tissue sample from a subject is to place the sample in a liquid that will suspend the metabolic activities of the cells. This process is commonly referred to as “fixation” and can be accomplished by several different types of liquids. The most common fixative in use by anatomical pathology labs is 10% neutral buffered formalin (NBF). This fixative forms cross-links between formaldehyde molecules and amine containing cellular molecules. In addition, this type of fixative preserves proteins for storage.
Another type of common fixative is ethanol or solvent based solutions. These fixatives tend to dehydrate the tissue and are commonly termed “precipitive fixatives.” As the term suggests, these solutions tend to denature proteins and inactivate cellular constituents in a manner different from formalin.
Biological samples that are “fixed” in 10% neutral buffered formalin preserve the tissue from autocatalytic destruction by cross-linking much of the protein and nucleic acids via methylene bridges. The cross-linking preserves the characteristics of the tissue, such as the tissue structure, cell structure and molecular integrity. Typically, fixation with 10% NBF takes several hours and can be thought of as two separate steps. First is the diffusion step where a large volume of formalin on the outside of the tissue needs to diffuse into the tissue. This process is governed by the laws of physics and depends on the tissue thickness, concentration of formalin and temperature (e.g., formalin temperature, tissue temperature, etc.). In the second step, the formalin molecules interact with biological molecules in the tissue, becoming incorporated into the methylene cross-links. This cross-link structure can keep the cellular structure intact during subsequent processing such as tissue dehydration and embedding the tissue into paraffin wax.
If the tissue is over-fixed, it may be difficult to diffuse processing liquids through the tissue due to the extensive network of cross-linked molecules. This can result in inadequate penetration of the processing liquids. If the processing liquid is a stain, slow diffusion rates can cause uneven and inconsistent staining. These types of problems can be increased if the stain has relatively large molecules. For example, conjugated biomolecules (antibody or DNA probe molecules) can be relatively large, often having a mass of several hundred kilodaltons, causing them to diffuse slowly into solid tissue with typical times for sufficient diffusion being in a range of several minutes to a few hours.
If the tissue is under-fixed, the tissue may be susceptible to severe morphology problems or autocatalytic destruction. Severe morphology problems result from an incomplete network of cross-linked molecules and subsequent shrinking of cells, nuclei and cytoplasm during dehydration steps. Autocatalytic destruction can result in loss of tissue structure, cell structure and tissue morphology, especially if the tissue is not processed within a relatively short period of time. Accordingly, under-fixed tissue may be unsuitable for examination and is often discarded.
To prepare biological samples for examination, tissues are often stained by using a variety of dyes, immunohistochemical (IHC) staining processes, or in situ hybridization (ISH). The rate of immunohistochemical and in situ hybridization staining of fixed tissue (e.g., paraffin embedded sectioned fixed tissue) on a microscope slide is limited by the speed at which molecules (e.g., conjugating biomolecules) can diffuse into the fixed tissue and interact molecularly from an aqueous solution placed in direct contact with the tissue section. In some tissues, such as relatively fatty tissue (e.g., breast tissue), it is difficult to predict fixation processing times due to these inaccessibility issues. Accordingly, tissues can be over-fixed (e.g., excessively cross-linked) or under-fixed (e.g., insufficiently cross-linked).
A wide variety of techniques can be used to analyze biological samples either prior to or after exposure to a fixative. Example techniques include microscopy, microarray analyses (e.g., protein and nucleic acid microarray analyses), mass spectrometric methods and a variety of molecular biology techniques. However, there are no suitable methods to determine the fixation state of a sample.
Conventional pathology practice is often based on predetermined fixation settings based on empirical knowledge of processing times for sample dimensions (e.g., thicknesses) and tissue type. It is often difficult to stain tissue without knowing this information; tissue is thus often tested to obtain such information. Unfortunately, the testing may be time-consuming, destroy significant portions of sample, and lead to reagent waste. By way of example, numerous iterations with different antigen retrieval settings for IHC/ISH stains may be performed in order to match and/or compensate for an unknown fixation state and an unknown tissue composition. The repeated staining runs result in additional sample material consumption and lengthy periods for diagnosis.
Acoustical energy has been used in a number of applications in science and medicine. These include attempts to speed up biological reactions ranging from assays that have molecular interactions to fixation of tissue samples. In addition, acoustics have long been used to monitor for the presence of submarines and other maritime vessels by the US navy. Acoustics have also been applied in monitoring ocean temperatures by measuring the speed of a signal between two points. Unfortunately, acoustics have not been used to determine desired characteristics of specimens.